Field of the Invention
An object of the invention pertains to a biocomposite for the use in medicine and veterinary to restore injured tissue and organs in mammalians, to a kit for making the biocomposite, a method of making the biocomposite, a method of treating inquiries, and a method of delivering nucleic acids.
Discussion of the Background
Known methods of tissue and organ restoration are based on engineered tissue constructions that contain either different cell populations or various genetic constructions (nucleic acids). Although both trends—cellular and genetic—are promising, they have moderate and limited efficacy.
A method according to which only a genetic construction is administered with ultrasound is, e.g., described in (Greenleaf W. J., Bolander M. E., Sarkar G. et al. Artificial cavitation nuclei significantly enhance acoustically induced cell transfection. Ultrasound in Medicine and Biology 1998; 24(4): 587-595; Schratzberger P., Krainin J. G., Schratzberger G. et al. Transcutaneous ultrasound augments naked DNA transfection of skeletalmuscle. Molecular Therapy 2002, 6(5): 576-583). However, the efficiency of administration of genetic constructions is low when parameters of ultrasonic radiation safe for a recipient are used. The delivery of the necessary amount of genetic constructions is possible only with the ultrasonic exposure damaging to tissues (see e.g., Duvshani-Eshet M., Machluf M. Therapeutic ultrasound optimization for gene delivery: a key factor achieving nuclear DNA localization. Journal of Controlled Release 2005; 108(2-3): 513-528; Kim H. J., Greenleaf J. F., Kinnick R. R. Ultrasound-mediated transfection of mammalian cells. Human Gene Therapy 1996; 7(11): 1339-1346).
Another method according to which nucleic acids are administered in a complex with liposomes is, e.g., described in (Fraley R., Subramani S., Berg P. et al. Introduction of liposome-encapsulated SV40 DNA into cells. J Biol Chem. 1980; 255(21): 10431-5) and a modified method, i.e., the administration of a combination of a liposome conjugate with genetic constructions using physical factors (e.g., ultrasound) is, e.g., described in (Roos A. K., Eriksson F., Timmons J. A., Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment. PLoS One. 2009; 4(9): e7226). However, a significant proportion of liposomes is destroyed when exposed to lipolytic enzymes in tissue which reduce the efficiency of delivery of active substances such as nucleic acids. The remaining part of nucleic acids is delivered almost concomitantly which does not allow achieving a prolonged effect. Besides, when transported into target cells, a complex “liposome—genetic construction” is not destroyed but rather lyposomes enter the cells which reduces the method safety.
A method according to which nucleic acids are administered via electroporation is, e.g., described in (Roos A. K., Eriksson F., Timmons J. A., Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment. PLoS One. 2009; 4(9): e7226; Schertzer J. D., Lynch G. S. Plasmid-based gene transfer in mouse skeletal muscle by electroporation. Methods Mol Biol. 2008; 433: 115-25). However, this method is based on the application of electric field which pushes genetic constructions (alone or in a complex with other components, e.g., an adjuvant) through a biological membrane. As a result of this method, a significant portion of genetic constructions is damaged. This method does not allow achieving the prolonged effect and, therefore, it is necessity to use the method repeatedly.
A method of locally administering an engineered tissue construction which includes a three-dimensional scaffold and cells adhered to its surface is, e.g., described in (Deev R. V., Tsipkina N. V., Bozo I. Ya., Pinaeva G. P., Tuhiliv R. M., A method of combining cultured osteogenic cells and a three-dimensional matrix-carrier, RU N22008150694, issued Dec. 22, 2008). However, in this set up, the cells require an active blood supply. A critical distance from the hemomicrocirculatory bloodstream, beyond which cells inevitably die, is 200-500 μM (see e.g., Polykandriotis E., Arkudas A., Horch R. et al. Autonomously vascularized cellular constructs in tissue engineering: opening a new perspective for biomedical science. J. Cell Mol. Med. 2007; 11(1): 6-20). Due to this, the size of a tissue-engineered construction, at which the cells of the construction after the transplantation into a recipient remain viable, should be not less 1 cm3.
Defects of a such size do not require additional optimization of the regenerative process, therefore tissue engineering constructions of a significantly larger size can be used (from 1 cm3). However, transplantation of a such large construction may damage a tissue area and may inevitably result in death of a larger proportion of the administered cells (especially the cells located in a central part of the biocomposite) which significantly reduces the efficiency of the method. The effectiveness of a tissue engineering construction in repairing a bone defect (or other applications) mainly depends upon survival of the cells which are comprised in a biocomposite. However, the cells of the tissue engineering construction need oxygenation and, therefore, the bigger tissue engineering construction, the more cells may die after transplantation (especially, the cells located in the central part (closer to the “nucleus”) of the tissue engineering construction). Thus, in large tissue engineering constructions (bigger than 1 cm3) many included cells may die and the efficiency of the repair may not be high (e.g., the efficiency may be the same as that of the scaffold without the cells). Therefore, small size tissue engineering constructions are more effective for bone replacement compared to the same scaffold without the cells. This problem of oxygenation is well-known.
In addition, a choice of a size of a tissue engineering construction is determined by the size of a bone defect which has to be repaired; the size of a tissue engineering construction has to match to the size of the bone defect. Thus, small tissue engineering constructions for repair of large defects may not be effectively used. Natural regeneration of small bone defects is quite good and does not require highly effective osteoplastic materials such as tissue engineering constructions. In this case, the regeneration is effective because most of the included cells are alive and perform their function. Thus, tissue engineering constructions are preferably used to repair large bone defects; however, in this case, one have to use tissue engineering constructions of a big size, and the majority of their cells may die and, therefore, the final efficiency becomes lower.
An activated matrix, a method of making the matrix, and local administration of nucleic acids into the matrix of a biocompatible made of various materials to provide reparative processes are, e.g., described in (Goldstein S. A., Methods of in vivo gene transfection for reparative wound regeneration, RU No22170104, issued Jun. 10, 2001). However, these technical approaches are related to a two-component product which includes a scaffold and a genetic construction. In this method, a cellular element which is required for the optimal histotypical restoration of tissue and organs, is absent and, therefore, the efficiency of the product application is moderate.
Thus, various approaches exist for making a biocomposite that provides reparative regeneration of tissue and organs.
One known approach is to combine a scaffold and cells (tissue engineering approach) in a biocomposite. However, when the complex contains a large volume of the biocomposite (greater than 1 cm3), the cells included in the biocomposite after transplantation into a recipient die because of the insufficient blood stream supply. The larger is a biocomposite, the more cells included in the biocomposite and located inside the scaffold die. Only cells located at the periphery survive. This problem of a blood supply of tissue-engineered biocomposites exists and has not been solved up to now.
Another known approach is to combine a scaffold and nucleic acids. However, the “safest” nucleic acids (i.e., nucleic acids carried by a non-viral delivery system, e.g., a plasmid) have a low level of transfection efficiency in vivo, i.e., only from 2 to 5% of the nucleic acids after the implantation of this biocomposite into a recipient transfect the cells. Nucleic acids included in viral delivery systems provide a higher level of transfection (e.g., the efficiency of transfection is up to 40-45%); however, virus-based systems are less “safe”, as they are introduced via viral particles (e.g., retrovirus, adenovirus, or lentivirus).
Thus, there is a need in a biological composite which lacks the deficiencies of known methods.